ABSTRACT The gastrointestinal pathogen enteropathogenic E. coli (EPEC) causes diarrhea and is responsible for significant morbidity and mortality in children under five years of age in developing countries. There are key gaps in our understanding of how EPEC causes diarrhea. The bacteria use a syringe-like type III secretion system to deliver key virulence ?effectors? directly into host intestinal epithelial cells. In the initial phase of infection, EPEC delivers proteins that promote host cell survival. Later in infection, however, secretion of more cytotoxic proteins results in host cell death. The studies proposed in this application focus on two EPEC effector molecules that are secreted at discrete times post-infection, and that have contrasting impacts on mitochondria, and on host cell survival. We hypothesize that the orchestrated and intersecting actions of effector proteins on host mitochondrial stability and function are critical for EPEC colonization and virulence. This hypothesis will be tested in three Aims where we will: (1) Mechanistically define the impact of two EPEC effectors on host mitochondrial structure, (2) Characterize effector-dependent alterations in mitochondrial function in infected intestinal epithelial cells in vitro, and (3) Establish the impact of EPEC secreted virulence effectors on mitochondrial function in vivo using a rabbit model of infection. The proposed studies will establish the precise role of key secreted virulence factors, and of host mitochondrial perturbations, in EPEC virulence. In the future, we anticipate our studies to inform the development and use of host cell-targeted molecules as adjuncts to standard therapies for treating the diarrhea caused by EPEC and related bacteria.